1. Field of the Invention
The present invention relates to disease detection and treatment for diseases such as cardiovascular and immune diseases and, more specifically, to a method for using lymphocyte activation gene 3 (LAG-3) profiling as a biomarker for assessing inflammasomes, chronic inflammatory diseases and dysfunctional HDL.
2. Description of the Background
The lipoprotein receptor, scavenger receptor class B type 1 (SR-BI), is a physiologically relevant receptor that modulates cholesterol levels, especially HDL-cholesterol (HDL-C), in mice and humans. In co-pending U.S. application Ser. No. 13/707,256 filed Dec. 6, 2012, the present inventor disclosed a method of genotyping women in order to identify the presence of the rs10846744 mutation of the SCARB1 gene (located on chromosome 12:q24.32). This was significantly associated with infertility, as well as subclinical atherosclerosis (SCA) and incident cardiovascular disease (CVD) in male participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Specifically, carriers of the risk C allele had significantly increased odds for incident CVD, and in a multivariate regression model this relationship was not attenuated by inclusion of traditional CVD risk factors such as age, body mass index, hypertension, smoking, renal disease, or lipid levels (whether total cholesterol, LDL-cholesterol [LDL-C], HDL-C, or triglycerides). These findings strongly suggested that other factors or pathways might be causal in the association of this genetic variant with incident CVD.
Interestingly, rs10846744 resides within the first intron of SCARB1 and bioinformatic analysis revealed that this single nucleotide polymorphism (SNP) resides within a regulatory region. The data suggested that this SNP could transcriptionally regulate genes on the same chromosome (intra-chromosomal) or inter-chromosomally. The present inventor investigated this possibility and a number of transcriptionally regulated gene candidates emerged. One in particular, lymphocyte activation gene-3 (LAG-3) is also located on chromosome 12 and was investigated further.
In vitro and ex vivo approaches were taken to examine the association of rs10846744 with LAG-3 in biospecimens isolated from hyperalphalipoproteinemic (HALP) women and men subjects. It was found that rs10846744 is significantly associated with alterations in the expression and function of LAG-3, and markers of intracellular inflammasomes.
LAG-3 is located near the CD4 loci on chromosome 12 (chr 12:p13) while rs10846744 is located on chr12:q24.32. LAG-3 has a similar function, if not a competitive one against CD4, by binding MHC class II on antigen presenting cells. [Sierro S, Romero P, Speiser D E. The CD4-like molecule LAG-3, biology and therapeutic applications, Expert Opin Ther Targets 2011; 15:91-101] in murine cells. Kisielow et al reported that activated T cells induced LAG-3 expression on B cells. [Kisielow M, Kisielow J, Capoferri-Sollami G, Karjalainen K. Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells. Eur J Immunol 2005; 35:2081-2088] They determined that LAG-3 induction on B cells was T cell dependent and not dependent on other stimuli such as unmethylated CpG motif 1826, bacterial LPS, or anti-Ig antibody in combination with anti-CD40 and IL-4. In contrast, LAG-3 RNA and protein was detected in EBV-transformed B cells, with significantly higher expression in EBV-transformed cells expressing the reference G allele as compared with cells expressing the risk C allele. Although EBV transformation of B lymphocytes could activate the cells, there was a significant difference in the level of LAG-3 expression based on rs10846744 genotype stratification. Importantly, others observed a lack of LAG-3 expression in B cells, such as Ramos cells [Baixeras E, Huard B, Miossec C, Jitsukawa S, Martin M, Hercend T, Auffray C, Triebel F, Piatier-Tonneau D., Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens, J. Exp. Med. 1992; 176:327-337] However, the present inventor genotyped these cells and found that they were heterozygous for the rs10846744 variant. More recently, Morales et al. showed that EBV positivity in Hodgkin lymphomas were significantly associated with increased gene expression of LAG-3. [Morales O, Mrizak D, Francois V, Mustapha R, Miroux C, Depil S, Decouvelaere A V, Lionne-Huyghe P, Auriault C, de Launoit Y, Pancre V, Delhem N. Epstein-Barr virus infection induces an increase of T regulatory type 1 cells in Hodgkin lymphoma patients. Br J Haematol 2014 Jul. 9, Epub ahead of print]
Baixeras et al., supra, characterized the cellular distribution of LAG-3 in a number of cell lines and demonstrated that LAG-3 resided within lipid rafts. Subsequently, Woo et al. reported the intracellular distribution of LAG-3 and found that LAG-3 was equally distributed between intracellular compartments and the plasma membrane. [Woo S-R, Li N, Bruno T C, Forbes K, Brown S, Workman C, Drake C G, Vignali D A A. Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4. Eur. J. Immunol 2010; 40:1768-1777] By using flow cytometry, the present inventor confirmed that low levels of LAG-3 were detected on the cell surface of risk C expressing cells regardless of stimulation conditions. However, LAG-3 was expressed on the cell surface in unstimulated reference G cells and its levels decreased significantly after stimulation, due to cleavage of cell surface LAG-3 by metalloproteases. These results in EBV transformed B cells are in contrast to those reported by Woo et al., supra, in that they reported that LAG-3 was expressed on the surface only in activated T cells.
It is also known that lipid raft signaling is essential for B cell activation. [Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 2000; 1:31-39] Specifically, stimulation of the B cell receptor (BCR) initiates phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of CD79A and CD79B (transmembrane immunoglobulin (Ig) receptor associated with Ig-alpha/Ig-beta heterodimers). [Schamel W W, Reth M. Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity. 2000; 13:5-14] Phosphorylation of ITAMs serve as docking sites for Syk, which is mediated by different Src family kinases (SFKs) including Fyn, Blk, and Lyn. [Takata M, Sabe H, Hata A, Inazu T, Homma Y, Nukada T, Yamamara H, Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 1994; 13:1341-9] Lyn is the major protein involved in lipid raft signaling upon B cell activation [Simons, supra]. This activation initiates the coordinate assembly of the “signalosome”, composed of a variety of intracellular signaling molecules and includes Btk, phosphatidylinositol 3-kinase (PI3K) and PLCγ2. [Blix E S, Irish J M, Husebekk A, Delabie J, Forfang L, Tierens A M, Myklebust J H, Kolstad A. Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma. BMC Cancer 2012; 12:478] PLCγ2 is the predominant isoform expressed in human B lymphocytes. [Coggeshall K M, McHugh J C, Altman A. Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in B lymphocytes. Proc Natl Acad Sci USA. 1992; 89:5660-4] It is also indispensable for BCR-mediated phosphoinositol hydrolysis and the subsequent biochemical events including PKC activation. [Sugawara H, Kurosaki M, Takata M, Kurosaki T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 1997; 16:3078-88]
However, the major apolipoprotein associated with HDL particles, apoA-I, has been shown to inhibit inflammatory cytokine production by inhibiting activation of monocytes by T lymphocytes. [Hyka N, Dayer J-M, Modoux C, Kohne T, Edwards III C K, Roux-Lombard P, Burger D. Apolipoprotein A-I inhibits the production of interleukin-1β and tumor necrosis factor-α by blocking contact-mediated activation of monocytes by T lymphocytes. Blood 2001; 97:2381-2389] Specifically, Hyka et al. observed that apoA-I inhibited cytokine production from stimulated monocytes by first binding to a surface factor, which suggests the possibility that apoA-I might interact with surface LAG-3.
The significant association of SCARB1 variant, rs10846744, with coronary heart disease (CHD) was shown in Manichaikul et al. [Arterioscler Thromb Vasc Biol 2012; 32:1991-1999]. However, previous analyses do not show that rs10846744 is directly associated with SCA and incident CVD. This is because, as the present inventor has found, LAG-3 is an important immune regulator that mediates the association of rs10846744 with atherosclerotic disease and CVD. Based on that mediator role that LAG-3 plays in CVD and other chronic inflammatory diseases, a method is herein disclosed for using LAG-3 expression profiling as a biomarker for assessing inflammasomes, chronic inflammatory diseases and dysfunctional HDL.